Journal: Protein Sci. / Year: 2017Title: Structure of the SLC4 transporter Bor1p in an inward-facing conformation.Authors: Nicolas Coudray / Sean L Seyler / Ralph Lasala / Zhening Zhang / Kathy M Clark / Mark E Dumont / Alexis Rohou / Oliver Beckstein / David L StokesAbstract: Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate ...Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid-Polyamine-organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Å resolution using cryo-electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo-EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all-atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward-facing conformation. Comparisons of the resulting Bor1p model with the X-ray structure of AE1 in an outward-facing conformation, together with MD simulations of inward-facing and outward-facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking-bundle transport mechanism described for other members of the APC superfamily.

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Image acquisition

Image acquisition

Number of digital images: 252

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Image processing

Processing

Method: helical reconstruction

3D reconstruction

Software: FREALIX, SPARX/EMAN2 / Resolution: 5.9 Å / Resolution method: FSC 0.143 CUT-OFFDetails: Reconstruction was done using D1 symmetry because of an existing two-fold symmetry in the unit cells composing the helical lattice. This two-fold axis runs perpendicular to the helical axis. For the final structure, a filter was applied to the map in order to compensate for resolution-dependent amplitude falloff. To do so, we built a model by arranging UraA in a helical assembly in order to mimic the mass distribution in Bor1p tubes. Fourier transforms from this model and from the experimental maps were then rotationally averaged to produce 1D scattering profiles. The resolution-dependent amplitude ratio from these profiles was used as a filter that was applied to the experimental amplitudes using SPARX routines. Finally, a low-pass filter was applied with a 5 Angstrom stop-band frequency.

FSC plot(resolution estimation)

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Atomic model buiding

Modeling #1

Refinement protocol: flexible / Target criteria: cross-correlation coefficient / Refinement space: REALDetails: A homology model was first created using human AE1 (PDB entry 4YZF) as a template using MODELLER, placed into the density map using SITUS, and then fitted into the map using the Molecular Dynamics Flexible Fitting (MDFF) method.Input PDB model: 4YZF

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Jul 12, 2017. Major update of PDB

Major update of PDB

wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary. This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated. See below links for details.

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